Broad range total available chlorine test strip

ABSTRACT

A composition, method, and test device for determining the total available chlorine concentration, and the relative amounts of free and bound chlorine, of a test sample are disclosed. The test device includes a test pad having a suitable carrier matrix incorporating an indicator reagent composition capable of converting combined available chlorine to free available chlorine and of interacting with free available chlorine to produce a detectable and measurable response for total available chlorine over a range of 0 to over 5000 ppm total available chlorine in the test sample. An indicator reagent composition contains: (a) an indicator dye that is responsive to free available chlorine, such as tetramethylbenzidine, (b) a buffer, (c) a surfactant, (d) an optional catalyst, and (e) an optional polymer. An indicator reagent composition is incorporated into a carrier matrix, like filter paper, to provide a test pad useful in a dry phase total available chlorine assay of a test sample, such as a sanitizing solution for a hemodialysis unit.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part application of U.S. application Ser. No.08/822,570, filed Mar. 19, 1997.

FIELD OF THE INVENTION

The present invention relates to a composition, method, and test devicefor determining the total available chlorine concentration of a testsample. More particularly, the present invention relates to a method andtest device for assaying a liquid test sample, such as a sanitizingsolution, for total available chlorine concentration over the range of 0to greater than 5000 ppm total available chlorine by using an improvedindicator reagent composition. The indicator reagent compositionundergoes a detectable and measurable response upon contact with a testsample containing free available chlorine. Contrary to priorcompositions and methods, the present method has the advantages ofmeasuring a wide range of total available chlorine and determining therelative amounts of free and bound chlorine in the test sample.

BACKGROUND OF THE INVENTION

The use of chlorine as a sanitizer or disinfectant for various watersupplies and various types of equipment, like food processing equipmentand medical equipment, such as a hemodialysis unit, is common. Becausethe amount of available chlorine in an aqueous solution relates directlyto the disinfecting or sanitizing activity thereof, a test which rapidlyand accurately measures available chlorine is important.

The available chlorine family is comprised of compounds which, when inaqueous solution, yield solutions of hypochlorous acid. The availablechlorine family is further divided into compounds containing freeavailable chlorine and compounds containing combined available chlorine.The sum of free available chlorine and combined available chlorine istermed total available chlorine.

Free available chlorine encompasses chlorine-containing compounds inaqueous solution such as hypochlorous acid, hypochlorite ion, and, instrong acid solutions, free chlorine. The use of free available chlorineas a disinfectant for water supplies and equipment is widespread becauseof its low cost, convenience, and effectiveness as an antiseptic agentin relatively low concentrations. For example, free available chlorineis used as a disinfectant in a majority of hemodialysis centers.

Combined available chlorine, also termed bound available chlorine,mainly encompasses organic chloramines, which release only a smallamount of free available chlorine in aqueous solution. Chloramines areformed from chlorine reacting with amine compounds in water. The aminecompounds can be an impurity in the water or arise from ammonia added towater with chlorine during water disinfection. Ammonia and chlorine areadded to the water to form chloramines which stabilize chlorine fromdecomposition and/or evaporation, and also increases the bacteriocidalpotency of chlorine. Depending on the ratio of chlorine-to-ammonia andthe acidity of the water, chloramines formed from chlorine and ammoniaare a mixture of monochloramine, dichloramine, and trichloramine atvarious ratios. Although monochloramine is the main chloramine ofconcern due to its toxicity, removal of all chlorine is essential forsafe and effective operation of a dialysis water purification system.

Conventionally, combined available chlorine has not been considered aneffective disinfectant or sanitizer. Accordingly, prior chlorine assayshave focused on assays for free available chlorine, i.e., the activedisinfectant. For example, assays disclosed in Rupe et al. U.S. Pat. No.4,092,115 and Ramana et al. U.S. Pat. No. 5,491,094, consider combinedavailable chlorine as an interferant in the assay for free availablechlorine, and the assays have been designed only to measure freeavailable chlorine. However, in some applications, it is important toassay for total available chlorine.

For example, chlorine is used in hemodialysis centers to sanitizehemodialysis units because chlorine is an effective and economicalsanitizing agent. It is important to clean and disinfect a hemodialysisunit between each dialysis session to prevent pathogen contaminationfrom patient to patient. However, chlorine also is a very toxic compoundthat can cause hemolysis even when only a trace amount of chlorinediffuses from the hemodialysis unit into the blood of an individual.Therefore, if an assay for residual chlorine in a hemodialysis unitdetects only free available chlorine, a potentially toxic amount ofcombined available chlorine, which slowly generates free availablechlorine, can be present to adversely affect an individual subsequentlyconnected to the hemodialysis unit. Trace amounts of free availablechlorine also can adversely affect filtration membranes of thehemodialysis unit.

Combined available chlorine is considered highly toxic because of itselectronic neutrality and ability to penetrate cell membranes. In amunicipal source water, combined available chlorine always exists invarious proportions relative to total available chlorine. Combinedavailable chlorine is formed in a reaction of free available chlorineeither with amine compounds, which are present as contaminants in thesource water, or with ammonia, which is added to the water with freechlorine to stabilize the chlorine and to increase the bacteriocidalpotency of the chlorine disinfectant.

In a dialysis unit, all chlorine species are removed before the watercan be used in hemodialysis. Chlorine removal is usually performed bypassing the water through a water purification tank containing activatedcarbon, and then through a reverse osmosis column. The presence ofcombined available chlorine in the water affects the efficacy of thecarbon tank in removing all chlorine species. Knowledge of theconcentration and the relative amount of combined available chlorine tototal chlorine is important in designing the water purification system,as well as devising a method of monitoring chlorine in the purifiedwater.

Occasionally, a trace amount of chlorine leaks through the tank. If thechlorine leaking through the tank is all, or substantially, combinedchlorine, this suggests exhaustion of carbon tank capacity. However, ifthe chlorine leaking through the tank contains a high proportion of freechlorine, this indicates the presence of a mechanical defect, such aschannelling through the activated carbon inside the tank. Determinationof both the free and combined available chlorine is important inmanaging the water purification for dialysis.

Therefore, when a sanitizing solution is used in medical or foodprocessing equipment, two critical chlorine levels must be monitored.First, the free available chlorine concentration must be sufficientlyhigh to perform a sanitizing or disinfecting function, i.e., at leastabout 1000 ppm (parts per million) free available chlorine is needed toeffectively sanitize equipment. Typically, a chlorine concentrationsufficient for equipment sanitization is about a 1 to 10 volume dilutionof a 5.25% (by weight) sodium hypochlorite with water, to provide asolution containing about 0.5% to about 0.6% (by weight) sodiumhypochlorite, i.e., about 5000 to about 6000 ppm chlorine. During thesanitizing process, the sanitizing solution is assayed periodically toensure that sufficient free available chlorine is present to sanitizethe equipment.

After the sanitizing function is completed, and before use, theequipment is rinsed with water to flush residual chlorine from theequipment. The rinse water also is assayed for available chlorine toensure that the level of residual available chlorine is below themaximum allowable level, e.g., 0.5 ppm as recommended by the Associationof Advancement of Medical Instrumentation (AAMI). In practice, theresidual available chlorine concentration is essentially zero, or atleast below the lowest detectable levels of about 0.1 to about 0.2 ppm,i.e., equivalent to a 1 to 100,000 water dilution of 5.25% (by weight)sodium hypochlorite.

Presently, only two types of commercial assay systems are suitable forassaying hemodialysis units for available chlorine. One assay utilizestablets or dry powder, and the other utilizes dry chemistry dip strips.Each assay has advantages and disadvantages, and neither assay satisfiesthe different testing requirements needed for a hemodialysis unit.

The tablet method has good sensitivity (e.g., 0.1 ppm) and is lessexpensive per assay. However, the tablet method is more cumbersome toperform and requires more technician time. The dry chemistry test stripsusually are not as sensitive as the tablet method and can cost more pertest. Nevertheless, the strip test is very easy and convenient,particularly when operating a mobile hemodialysis unit. In mosthemodialysis centers, the test strip is used as a screening test forresidual chlorine, whereas the tablet method is used for more criticalwater testing. Because of the differences in test requirements, mosthemodialysis centers are forced to stock both the tablet and drychemistry test systems.

To date, no known single assay is available to assay both the high andthe low available chlorine concentration range because the 10,000 folddifference in chlorine concentration between a working sanitizingsolution and a residual chlorine concentration makes detection anddifferentiation of concentration levels difficult. The present inventionis directed to providing a single assay for total available chlorinethat is capable of measuring total available chlorine concentration overthe range of 0 to greater than 5000 ppm, and especially 0 to greaterthan 1000 ppm.

The present invention, therefore, is directed to an assay method anddevice that can be used to test: (a) a working sanitizing solution,containing 500 ppm or more of free available chlorine, without dilutingthe solution, and (b) residual chlorine, both free and bound availablechlorine, present at 0.5 ppm or less in rinse water. The presentinvention also is directed to a method of simultaneously determining therelative amounts of free and bound chlorine in a test sample containing0.5 ppm or less residual chlorine. Such a determination providesimportant information with respect to the effectiveness of a chlorinesolution and whether potentially harmful amounts of residual boundchlorine are present in rinse water.

Accordingly, a test strip can be used either to test for residualchlorine in the rinse water after cleaning the hemodialysis unit, or totest for the available chlorine content of a working solution. Asillustrated hereafter, the present test strips have a good sensitivityand a wide detection range with a continuous color response from 0.5 toover 1000 ppm total available chlorine.

The present method of assaying for total available chlorine in anaqueous test sample yields trustworthy and reproducible results byutilizing an indicator reagent composition that undergoes a colortransition in response to total available chlorine concentration, andnot as a result of a competing chemical or physical interaction, such asa preferential interaction with another test sample component. Forexample, the present indicator reagent composition has sufficientsensitivity to detect as little as 0.1 ppm total available chlorine.Additionally, the method and composition utilized in the total availablechlorine assay does not adversely affect or interfere with any othertest reagent pads that are present on a multiple test pad strip. As setforth hereafter, methods are available such that the composition can beused to determine the relative amounts of free and bound availablechlorine in a test sample.

In accordance with the present invention, an indicator reagentcomposition can be incorporated into a carrier matrix to providesufficient sensitivity and color differentiation to assay for totalavailable chlorine concentration over the range of 0 ppm to greater than5000 ppm, and typically greater than 1000 ppm. In addition, although dryphase test strips have been used to assay for chlorine concentration, orto assay for the relative amounts of free and bound available chlorinein a test sample, no dry phase test strip has been used to assay fortotal available chlorine over such a wide concentration range, examplesof prior disclosures relating to assaying for chlorine include StormU.S. Pat. No. 3,718,605; Reiss U.S. Pat. No. 4,938,926; Ross, Jr. et al.U.S. Pat. No. 4,049,382; Frant U.S. Pat. No. 5,300,442, Harp U.S. Pat.No. 5,362,650; O'Brien et al. U.S. Pat. No. 4,904,605; and J. D. Johnsonet al., Analytical Chemistry, 40(13), pages 1744-1750 (1969).

SUMMARY OF THE INVENTION

In brief, the present invention is directed to a new and improvedcomposition, test device, and method of determining the total availablechlorine concentration of a test sample, and the relative amounts offree and bound available chlorine in the test sample. A device includesa test pad comprising a suitable carrier matrix incorporating anindicator reagent composition capable of converting combined availablechlorine to free available chlorine, and interacting with free availablechlorine to produce a detectable response to total available chlorineconcentration. A carrier matrix of the test pad comprises a bibulousmaterial, such as filter paper; a nonbibulous material, such as a strip,layer, or membrane of a polymerized material; or a mixture thereof. Anindicator reagent composition is homogeneously incorporated into thecarrier matrix, and the carrier matrix then holds the indicator reagentcomposition homogeneously throughout the carrier matrix whilemaintaining carrier matrix penetrability by the test sample.

More particularly, the present invention is directed to a method ofassaying for the total available chlorine content of aqueous testsamples by utilizing a new indicator reagent composition. It has beendemonstrated that a reagent composition including: (a) an indicatorcapable of interacting with free available chlorine to provide adetectable and measurable response, (b) a buffer, like a polycarboxylicacid, (c) a surfactant, like an anionic surfactant, (d) an optionalcatalyst, and (e) an optional polymer, affords sufficient sensitivity totest sample total available chlorine content, and a sufficient colordifferentiation between test samples of different total availablechlorine content over the range of 0 to greater than 5000 ppm, andparticularly 0 to greater than 1000 ppm. In accordance with an importantfeature of the present invention, the indicator reagent composition hasa pH of about 4 to about 6, and in preferred embodiments contains acatalytic amount of iodide ion or a peroxidase enzyme.

In accordance with an important feature of the present invention, anaccurate and reliable quantitative determination for total availablechlorine in a test sample is achieved because the indicator reagentcomposition is maintained at a pH of about 4 to about 6, and optionallycontains an iodide ion or peroxidase catalyst. By utilizing an indicatorreagent composition of the present invention, the quantitative assay fortotal available chlorine in liquid test samples is more sensitive andaccurate because the combined available chlorine in the test sample isquickly converted to free available chlorine. The indicator reagentcomposition then is able to detect the total available chlorine presentin the test sample.

Therefore, a buffer is included in the indicator reagent composition tomaintain a pH of about 4 to about 6 and achieve a more accuratemeasurement of the total available chlorine concentration of the testsample. The buffer is included in the indicator reagent composition tomaintain the reagent composition within a pH range wherein the combinedavailable chlorine, i.e., chloramine, is quickly converted to freeavailable chlorine. The presence of a catalytic amount of iodide ion ora peroxidase enzyme further facilitates, and speeds, conversion ofcombined available chlorine to free available chlorine.

Therefore, one aspect of the present invention is to provide a methodand composition for quantitatively determining the total availablechlorine concentration of an aqueous liquid. The composition convertsthe combined available chlorine to free available chlorine, andinteracts with the free available chlorine to produce a change in colorof a test device that is indicative of the total available chlorineconcentration of the test sample.

Another aspect of the present invention is to provide a method ofassaying aqueous test samples, said method having sufficient sensitivityand sufficient visual color resolution to allow differentiation between,and the quantitative measurement of, test samples having different totalavailable chlorine concentrations.

Yet another object of the present invention is to provide a sensitivemethod of assaying test samples for total available chlorineconcentration over the range of 0 to greater than 5000 ppm totalavailable chlorine. Accordingly, the present method is able to detectresidual chlorine present in rinse water, i.e., less than 0.5 ppm, orworking concentration of free available chlorine above 1000 ppm.

Another aspect of the present invention is to provide a method ofassaying test samples to determine the relative amounts of free andbound available chlorine in the test sample.

Another aspect of the present invention is to provide an indicatorreagent composition that interacts with free available chlorine andundergoes a visually or instrumentally differentiable color transitionto allow the determination of total available chlorine concentration ofa test sample.

Another aspect of the present invention is to provide a method ofassaying for the total available chlorine content of a liquid testsample by incorporating an indicator reagent composition into a dryphase detection device, wherein the indicator reagent compositioncomprises: (a) an indicator capable of interacting with free availablechlorine to provide a detectable and measurable response, (b) a buffer,(c) a surfactant, (d) an optional catalyst, (e) an optional polymer, and(f) a suitable carrier, and wherein the indicator reagent compositionhas a pH of about 4 to about 6.

Still another aspect of the present invention is to provide a new andimproved method of assaying for the total available chlorine content ofan aqueous test sample by utilizing a test device, including a carriermatrix, said carrier matrix comprising a bibulous matrix, like filterpaper, or a nonbibulous matrix, like a glass fiber or a layer of apermeable polymeric material, and said carrier matrix havingincorporated therein an indicator reagent composition capable ofconverting bound available chlorine to free available chlorine, and ofinteracting with free available chlorine present in the test sample, toprovide a color transition that can be correlated to the total availablechlorine concentration of the test sample.

A further aspect of the present invention is to provide an improved dryphase test strip that incorporates an indicator reagent compositioncomprising a suitable indicator, a buffer, and a surfactant, and havinga pH of about 4 to about 6, into the carrier matrix, and thereby providea quantitative assay for the total available chlorine content of a testsample.

The above and other aspects and advantages and novel features of thepresent invention will become apparent from the following detaileddescription of the preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the detection pattern for free available chlorine ona test strip of the present invention;

FIG. 2 illustrates the detection pattern for bound available chlorine ona test strip of the present invention;

FIG. 3 illustrates the detection pattern for free and bound availablechlorine on a test strip of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the method of the present invention, a quantitativeassay of aqueous test samples for total available chlorine isaccomplished by utilizing an indicator reagent composition that includes(a) an indicator capable of interacting with free available chlorine toprovide a detectable and measurable response, (b) a buffer, (c) asurfactant, (d) an optional catalyst, and (e) an optional polymer. Byemploying an indicator reagent composition of the present invention,having a pH of about 4 to about 6, sufficient sensitivity and sufficientvisual color differentiation between test samples of different totalavailable chlorine content is achieved. In accordance with the method ofthe present invention, test samples having a total available chlorinecontent of 0 to greater than 5000 ppm, and particularly 0 to greaterthan 1000 ppm, can be measured and differentiated.

To achieve the full advantage of the present invention, the method andcomposition are employed in dry phase, test pad assays to determine thetotal available chlorine concentration of aqueous test samples. A dryphase test strip, including a test pad comprising a carrier matrixincorporating an indicator reagent composition of the present invention,allows the rapid quantitative assay of test samples by visual means.

In particular, the present invention allows determination of the totalavailable chlorine concentration of a test sample by the visual colorchange of a test pad on a test strip resulting from contact between thetest strip and the test sample. Total available chlorine concentrationof the test sample is determined by correlating the detected freeavailable chlorine concentration to the total available chlorineconcentration of the test sample. The test strip includes a test padcomprising an inert carrier matrix incorporating an indicator reagentcomposition. The present composition and method allow the rapidcolorimetric determination of the total available chlorine concentrationof a test sample by quickly converting the bound available chlorine tofree available chlorine, and assaying for the resulting total freechlorine concentration. Previous assay methods employed compositionsthat avoided measurement of bound available chlorine. In contrast, thepresent method measures the total available chlorine content, i.e., freeand bound available chlorine, by utilizing a composition having a pH ofabout 4 to about 6, and which optionally contains a catalyst to increasethe rate of conversion of combined available chlorine to free availablechlorine.

In accordance with an important feature of the present invention, thetest sample can be applied to the test strip in a manner that permits adetermination of the relative amounts of free and bound availablechlorine in the test sample. In this method, the free and boundavailable chlorine are independently, and simultaneously, detected in asingle test strip.

An important component of the present indicator reagent composition isthe indicator. The indicator included in the indicator reagentcomposition is limited only in that the indicator is capable ofundergoing a detectable response, and preferably a chromogenic response,in the presence of free available chlorine. Accordingly, the indicatorpreferably is a redox indicator that undergoes a color transition, orother detectable response, upon conversion from its reduced state to itsoxidized state by free available chlorine. The indicator dye should besufficiently stable such that free available chlorine is present beforea color transition occurs. To achieve the full advantage of the presentinvention, the indicator dye undergoes a color transition throughvarious detectable and measurable degrees and intensities of color suchthat the degree and intensity of the color transition can be correlatedto the concentration of total available chlorine in a test sample.

It should be noted that the indicator is incapable of interacting withbound available chlorine. Therefore, as explained in detail hereafter,the bound available chlorine is converted to free available chlorine.The indicator, therefore, responds to the total available chlorineconcentration of the test sample.

The indicator, therefore, typically is a redox indicator. Preferredredox indicators are the benzidine-type indicators, i.e., benzidine andbenzidine derivatives. The benzidine-type indicators have the ability todevelop easily detectable and differentiable color hues of varyingintensity, which makes these indicators useful in quantitative assays.Although the exact mechanism of color formation by benzidine-typeindicators in the presence of various analytes is not known, it is knownthat two sequential color forms occur: a first colored species which isblue in color, and a second colored species, which is brown. The bluecolor species tends to be transient and changes to the brown colorspecies. Therefore, it has been necessary to detect a color changewithin a prescribed time period or to stabilize the blue color.Otherwise, the significance of the color transition, i.e., correlationto analyte concentration, is lost because subtle shades of blue, whichare easily distinguishable, yield to the less easily differentiatedbrown hues.

Benzidine-type indicators have the structure: ##STR1## wherein the R¹and R² substituents, same or different, can be hydrogen, lower alkyl(i.e., alkyl having 1 to about 6 carbon atoms), lower alkyloxy (i.e.,alkyloxy having 1 to about 6 carbon atoms), amino, aryl, or aryloxy.Moreover, the R² substituents together can form .paren open-st.CH₂.paren close-st._(n), wherein n is 1 or 2. In addition to the above, theR¹ and R² groups can be substituted such as with hydroxy, halogen,cyano, and similar substituents. Typical benzidine-type indicatorsinclude, but are not limited to, benzidine, o-tolidine, o-dianisidine,2,7-diaminofluorene, 3,3',5,5'-tetramethylbenzidine (hereaftertetramethylbenzidine or TMB), 3,3'-diaminobenzidine,3,3',5,5'-tetra(alkyl)benzidine, the various N- and N'-substitutedbenzidines and others, and mixtures thereof.

Another useful class of dyes are the heterocyclic azine indicators, forexample, bis(N-ethylquinol-2-one)azine and (N-methylbenzthiozal-2-one)(1-ethyl-3-phenyl-5-methyltriazol-2-one)azine. Preferably, the indicatoris a benzidine-type indicator. To achieve the full advantage of thepresent invention, the indicator is 3,3',5,5'-tetramethylbenzidine.

The indicator typically is present in the indicator reagent compositionin a concentration of about 1 to about 200 mM, and preferably in aconcentration of about 10 to about 150 mM. The amount of indicator inthe indicator reagent composition can be less than about 1 mM, orgreater than about 200 mM, depending upon the intensity of the colortransition that a particular indicator undergoes upon oxidation. Ingeneral, the amount of indicator included in the indicator reagentcomposition is limited only in that the indicator undergoes a detectablecolor transition in proportion to the concentration of free availablechlorine. The detection of free available chlorine then can becorrelated to total available chlorine content of the test sample.

In addition to the indicator, the indicator reagent composition alsocontains a buffer. In accordance with an important feature of thepresent invention, the buffer buffers the indicator reagent compositionin the range of about 4 to about 6, and preferably about 4.5 to about5.5. To achieve the full advantage of present invention, the buffermaintains the composition at a pH of about 4.8 to about 5.3. In the pHrange of about 4 to about 6, the bound available chlorine, i.e.,chloramine, is converted to free available chlorine at a sufficient ratesuch that the bound available chlorine is assayed and detected by thepresent indicator reagent composition.

A pH of about 4 to about 6, therefore, provides an indicator reagentcomposition having a very high sensitivity to chlorine. Free availablechlorine interacts with an indicator over a wide pH range. However,bound available chlorine cannot interact with the indicator above pHabout 6. The interaction between bound available chlorine and theindicator increases significantly at pH about 5.5 or less, andespecially at pH 5 or less, i.e., bound available chlorine is convertedto free available chlorine, which in turn can interact with theindicator. However, below pH about 5, the development of a backgroundcolor increases. This background color interferes with an accurate assayfor total available chlorine. Therefore, to achieve the full advantageof the present invention, the indicator reagent composition is bufferedat a pH of about 4.8 to about 5.3.

The identity of the buffer is not particularly limited, as long as theindicator reagent composition is buffered in the range of about 4 toabout 6. Therefore, useful buffers include, but are not limited to,polycarboxylic acids, phosphate, borate, acetate, and mixtures thereof.Preferred buffers are polycarboxylic acids, and especiallypolycarboxylic acids wherein the carboxyl groups are separated by two tofive carbon atoms. Examples of useful polycarboxylic acid buffersinclude, but are not limited to, citric acid, succinic acid, lacticacid, and ketoglutaric acid. As demonstrated in detail hereafter, thepolycarboxylic acid buffers improve the color response of the indicatorto free available chlorine, and provide a more stable color response. Ithas been theorized, but is not relied upon herein, that thepolycarboxylic acid is capable of complexing with the indicator to forma brighter and more spectacular color, and to stabilize the color. Theconcentration of buffer in the composition typically is about 20 toabout 600 mM, and preferably about 50 to about 200 mM.

In addition to the indicator and the buffer, the indicator reagentcomposition also contains a surfactant, in particular an anionicsurfactant or a nonionic surfactant. The surfactant is present in theindicator reagent composition in an amount of about 0.05% to about 1.5%,and preferably about 0.1% to about 1%, by weight of the composition. Toachieve the full advantage of the present invention, the surfactant ispresent in an amount of about 0.15% to about 0.5% by weight of thecomposition.

As illustrated in detail hereafter, the surfactant not only improves theability of the test sample to wet the carrier matrix, but the surfactantalso improves the stability of the color transition of the indicator inresponse to free available chlorine. As further illustrated hereafter,the surfactant helps permit the present indicator reagent composition toassay for a broad range of total available chlorine, i.e., 0 ppm togreater than 5000 ppm.

With respect to wetting and color stabilization for test samplescontaining 0 to about 20 ppm total available chlorine, the surfactantcan be an anionic, or a nonionic, surfactant. Each of these classes ofsurfactants effectively stabilizes the light blue to blue color providedby the indicator. Cationic surfactants and zwltterionic surfactants, asdemonstrated hereafter, did not stabilize the color transition.

With respect to color stabilization for test samples containing greaterthan about 20 ppm total available chlorine, the surfactant typicallycomprises an anionic surfactant. Anionic surfactants allowed theindicator reagent composition to successfully assay test samples havinga total available chlorine concentration of 20 ppm or greater, up toabout 5000 ppm.

Useful nonionic surfactants include, but are not limited to, anethoxylated polysorbate, e.g., polysorbate 20 through polysorbate 85, anethoxylated alcohol, e.g., a C₁₀ to C₂₂ alcohol ethoxylated with about10 to about 25 moles of ethylene oxide, an ethoxylated phenol, i.e., anethoxylated octylphenol, nonylphenol, or dodecylphenol with about 8 toabout 30 moles of ethylene oxide, a polyethylene glycol, e.g., PEG-8through PEG-40, a polypropylene glycol, e.g., PPG-9 through PPG-34, anethylene glycol-propylene glycol copolymer, e.g., a poloxamer, apolybutylene glycol, and similar nonionic surfactants, and mixturesthereof. In general, a useful nonionic surfactant has an HLB value ofabout 6 to about 25.

Anionic surfactants useful in the present invention are not particularlylimited. Usually, the anionic surfactant includes a hydrophobic moiety,such as a carbon chain including about eight carbon atoms to about 30carbon atoms, and particularly about twelve carbon atoms to about twentycarbon atoms; and further includes a hydrophilic moiety, such assulfate, sulfonate, carbonate, phosphate, or carboxylate. Often, thehydrophobic carbon chain is etherified, such as with ethylene oxide orpropylene oxide, to impart a particular physical property or reducedsurface tension, to the anionic surfactant.

The anionic surfactants are well known, and can be a fatty acid, a saltof a fatty acid, an ethoxylated fatty acid, or a salt of an ethoxylatedfatty acid, for example. Therefore, suitable anionic surfactantsinclude, but are not limited to, compounds in the classes known as alkylsulfates, alkyl ether sulfates, alkyl ether sulfonates, sulfate estersof an alkylphenoxy polyoxyethylene ethanol, alpha-olefin sulfonaLes,beta-alkyloxy alkane sulfonates, alkyl arylsulfonates, alkyl carbonates,alkyl ether carboxylates, fatty acids, sulfosuccinates, alkyl ethersulfosuccinates, sarcosinates, octoxynol phosphates, nonoxynolphosphates, taurates, fatty taurides, sulfated monoglycerides, fattyacid amido polyoxyethylene sulfates, and isothienates; or mixturesthereof. Many additional anionic surfactants are described inMcCutcheon's, Detergents and Emulsifiers, 1993 Annual, published byMcCutcheon Division, MC Publishing Co., and incorporated herein byreference.

Usually, the anionic surfactant is present in the composition as aneutralized salt in the form of a sodium, potassium, lithium, ammonium,alkylammonium, or hydroxyalkylammonium salt, wherein the alkyl moietyincludes one to about three carbon atoms. The alkyl sulfates and alkylether sulfates are particularly effective classes of anionicsurfactants. Consequently, exemplary anionic surfactants useful in thecomposition and method of the present invention include, but are notlimited to, the ammonium, mongethanolamine, diethanolamine,triethanolamine, isopropylamine, sodium, potassium, lithium, ormagnesium salt of lauryl sulfate, dodecylbenzenesulfonate, laurylsulfosuccinate, lauryl ether sulfate, lauryl ether carboxylate, laurylsarcosinate, cocomethyl tauride, and sulfosuccinate half ester amide; ormixtures thereof. Examples of especially useful anionic surfactants area lauryl sulfate salt, a lauryl ether sulfate salt, a lauryl phosphate,a sulfosuccinate salt, a dodecylsulfonate salt, a cholate salt, a C₈ toC₁₈ fatty acid, and mixtures thereof.

In addition to the indicator, buffer, and surfactant, the indicatorreagent composition also optionally can contain 0 to about 1000 ppm of acatalyst to increase the rate at which the indicator reagent compositionconverts the combined available chlorine to free available chlorine,thereby making it possible to assay for the amount of combined and free,i.e., total, available chlorine in the test sample.

In one embodiment, the catalyst is a peroxidase, like horseradishperoxidase. The peroxidase catalyst is present in the indicator reagentcomposition in an amount of 0 to about 1000 ppm. The peroxidase reducesthe interference of ammonium ions in the total available chlorine assay.It is important to reduce ammonium ion interference because ammoniumions react with free available chlorine to form a chloramine, andthereby convert free available chlorine to bound available chlorine. Thepresence of a peroxidase reverses this reaction, and frees the boundavailable chlorine from chloramine such that the resulting freeavailable chlorine is available for assay by a present indicator reagentcomposition.

The finding that a peroxidase stabilizes and enhances color formation ina total available chlorine assay is both new and unexpected. It wasexpected that a peroxidase, which is a protein, would interfere in thereaction between free available chlorine and the indicator. Thisinterference is expected because free available chlorine reacts withnitrogen present in a protein to form a chloramine, which binds chlorineand makes it unavailable for reaction with the indicator. Surprisingly,however, it was found that incorporating a catalytic amount of aperoxidase into a present indicator reagent composition does notinterfere with the reaction between the indicator and free availablechlorine and thereby reduce test strip sensitivity, but, to thecontrary, increases the sensitivity of the test strips and yields anaccurate quantitative assay for total available chlorine.

In particular, the effect of incorporating horseradish peroxidase isillustrated below. Free available chlorine reacts with variety of redoxindicators, the most common of which are benzidine indicators, such asorthotolidine or 3,3',5,5'-tetra-methylbenzidine. An important problemassociated with such indicators is that they exhibit a very narrowsensitivity range for chlorine, i.e., there is no variation of teststrip color when chlorine level is 10 ppm or higher. Therefore, it isimpossible to visually quantify a chlorine level greater than about 10ppm. However, by adding horseradish peroxidase to an indicator reagentcomposition, the test strip color varies over the range 0 ppm to greaterthan 1000 ppm total available chlorine with a differentiable colortransition from colorless to light blue to dark blue to greenish brownto brown. Distinct color separation differentiation was observed forfree available chlorine levels of 0, 0.5, 1, 5, 10, 100, 300, 500, and1000 ppm.

In particular, two indicator reagent compositions were prepared andincorporated into individual test strips. The indicator reagentcompositions were identical, except one composition containedhorseradish peroxidase. When the peroxidase was absent, the test stripsturned brown when the free available chlorine content was 10 ppm orgreater. When the peroxidase was present, the test strips changed colorfrom very light blue to light blue, blue, darker blue, dark blue, blackblue, greenish brown, and brown in response to free available chlorinelevels of 0.5, 1, 5, 10, 100, 300, 500, 1000 ppm, respectively.

In another test, it was found that peroxidase increases the rate ofconversion of combined available chlorine to free available chlorine. Itwas found that a test strip incorporating an indicator reagentcomposition lacking peroxidase reacted very slowly with a test samplecontaining 1 ppm of chloramine. However, when peroxidase wasincorporated into the composition, a test strip reacted with 1 ppm ofchloramine in 45 seconds to generate a color transition identical to 1ppm of free available chlorine.

Incorporating a peroxidase into an indicator reagent compositionprovides another advantage. In particular, when peroxidase is present,the test strip can be used to assay for peroxide in addition to totalavailable chlorine. Hydrogen peroxide is a commonly used disinfectantfor hemodialysis units, but most indicator reagent compositions areinsensitive to peroxide unless a catalytic peroxidase enzyme is present.

Accordingly, a present test strip can be used to assay for peroxide overthe range of about 0.1 to about 400 ppm. This capability greatlyincreases versatility of the present test strips because medical workersoften use hydrogen peroxide to sanitize hemodialysis units. The presenttest strips, therefore, can be used by medical personnel as the soletest strip to assay for residual sanitizing compounds in the rinse waterof a hemodialysis unit, regardless of whether the sanitizer is chlorineor a peroxide. Medical laboratories and clinics, therefore, do not haveto stock different types of assay kits for testing low total availablechlorine, high total available chlorine, and peroxide. Similar totesting for low, residual amounts of chlorine, the present test stripsturn blue upon contact with a test sample containing residual peroxide.A single test strip, therefore, provides the convenience of a dual testsystem for hydrogen peroxide and total available chlorine.

In another embodiment, the catalyst is iodide ion, typically in the formof an alkali metal iodide, like lithium iodide, sodium iodide, or,preferably, potassium iodide. An excess amount of iodide ion caninterfere with the assay. Therefore, when iodide ion is used as thecatalyst, the amount of iodide ion present in the indicator reagentcomposition is 0 to about 20 ppm, and preferably 0 to about 10 ppm. Toachieve the full advantage of the present invention, the amount ofiodide ion present in the composition is 0 to about 5 ppm. It has beendetermined that as little as 1 ppm of iodide ion can react with at leastfour equivalents of chloramine, thereby increasing the rate ofconversion of bound available chlorine to a detectable free chlorinespecies.

Iodide ion allows an indirect assay of the bound available chlorine. Inparticular, the catalytic mechanism involves recycling of iodide ion andiodine. The iodide ion first is oxidized by chloramine to iodine, whichin turn oxidizes an indicator, like tetramethylbenzidine (TMB), to anoxidized, colored indicator complex. As a result, iodine is reduced backto iodide ion which then repeats the oxidation-reduction cycle until allof the chloramine substrate is consumed. Like any catalytic reaction,the rate of this conversion is proportional to the level of catalyst. Ata potassium iodide concentration of 0.5 ppm, a reaction to completelyconvert 5 ppm of chloramine takes about 50 seconds. At a potassiumiodide concentration of 2 ppm, however, completion of the reaction isalmost instantaneous. ##STR2##

In accordance with an important feature of the present invention, theamount of iodide ion, therefore, can be maintained at a very low levelto perform its intended function, and, at the same time, not interferewith the assay for total available chlorine. If the amount of iodide ionis too high, iodine can be formed too rapidly and precipitated beforethe iodine can react with the indicator. As a consequence, the intensityof color transition is reduced, and a low assay results. At the sametime, the yellow/brown color of iodine causes strip color to shift to adirty green hue, making color differentiation to correlate the colortransition to total available chlorine concentration more difficult.

An optional polymer also can be incorporated into the indicator reagentcomposition. The polymer improves the stability and uniformity of thecolor transition of the test device. The polymer also helps incorporatethe indicator reagent composition uniformly throughout the carriermatrix. Suitable polymers include, but are not limited to,polyvinylpyrrolidone, polyvinyl alcohol, gum arabic, gelatin, algin,carrageenan, casein, albumin, methyl. cellulose, and similar natural andsynthetic polymeric materials. Specific examples of natural,cellulose-type polymers are hydroxypropylcellulose,hydroxyethylcellulose, hydroxybutylcellulose, and sodiumcarboxymethylcellulose. A useful synthetic polymer is apolyvinylpyrrolidone of average molecular weight about 40,000 andavailable commercially from ISP Corp., Wayne, N.J. A polymer generallyis included in the indicator reagent composition in an amount rangingfrom 0% to about 4%, and preferably from about 0.2%; to about 3%, bytotal weight of the indicator reagent composition.

The natural, cellulose-type polymers are preferred over syntheticpolymers, like polyvinylpyrrolidone, because the synthetic polymers havea tendency to impart a green hue to the test strip. The cellulose-typepolymers impart a preferred bluish color. The polymers also serve athickening function to help facilitate impregnation of the carriermatrix with the indicator reagent composition.

The presence of a polymer in an indicator reagent composition alsoenhances chloramine reactivity. The effectiveness of a polymer withrespect to enhancing chloramine reactivity is directly related to thehydrophobicity of the polymer. For example, GANTREZ ES225 (an ethylester of a PVM/NA copolymer), KLUCEL (hydroxypropylcellulose), and PVPK60 (polyvinylpyrrolidone) have a decreasing effectiveness with respectto enhancing chloramine reactivity, and also have a decreasinghydrophobicity. Test strips impregnated with an indicator reagentcomposition containing GANTREZ ES225 were essentially 100% reactive withchloramine, but also tended to develop background color withchlorine-free water. PVP K60 generated little background color, but isonly 50% reactive with chloramine. KLUCEL provided the best overallresults when considering both chloramine reactivity and lack ofbackground color. In particular, when using KLUCEL, the test strip hadan 80% to 90% chloramine reactivity and no background color.

The above-mentioned chloramine reactivities were evaluated by comparingthe color of a test strip after immersion into hypochlorite (freechlorine) solutions either containing or free of ammonium sulfate. Theaddition of ammonium ions to the hypochlorite solution convertshypochlorite to chloramine, which consequently is unreactive to freechlorine test strips. Test strip reactivity to chloramine can beimproved and restored to 100% by various approaches described herein,including a pH of about 4 to 6, the presence of a surfactant, thepresence of a catalyst, and/or the presence of a polymer. The percentreactivity was visually estimated by comparing the intensity of teststrip color with chloramine in relationship to the equivalent test stripcolor with free chlorine.

In addition, if necessary or if desired, inert background dyes can beincluded in the reagent composition to improve the color resolution anddifferentiation of the color transition in the present assay for totalavailable chlorine. Suitable background dyes include, but are notlimited to, ethyl orange (4-(4-diethylaminophenylazo)benzenesulfonicacid); Orange G (4-(2-hydroxy-(7,9 sodiumdisulfonate)-1-naphthylazo)-benzene); disperse orange 11, 13, or 25;calcomine orange; methyl orange; and orange II(4-(2-hydroxy-1-naphthylazo) benzenesulfonic acid), or mixtures thereof.A background dye is included in an indicator reagent composition of thepresent invention in a concentration of 0 mM to about 2 mM, andpreferably 0 mM to about 1 mM.

The carrier for the ingredients of an indicator reagent compositionincludes water. However, because of the limited water solubility ofparticular ingredients included in the indicator reagent composition,organic solvents, such as acetone, methanol, ethanol, isopropyl alcohol,ethylene glycol, propylene glycol, dimethylformamide, dimethylsulfoxide,acetonitrile, ethyl acetate, and similar solvents can be included in thecarrier vehicle. The selection of a suitable organic solvent orsolvents, in addition to water, to include in the carrier of theindicator reagent composition is within the capability of those skilledin the art of designing diagnostic assays.

The amount of organic solvent present in an indicator reagentcomposition generally is 0%, to about 90%, and preferably about 10%, toabout 70%, by weight of the carrier. A carrier comprising water and anorganic solvent, like methanol, ethanol, or acetone, is especiallypreferred because a carrier matrix impregnated with the indicatorreagent composition can be dried within a few to several minutes.

As previously described, an indicator reagent composition undergoes acolor transition upon contact with a test sample to provide an assay fortotal available chlorine concentration from the intensity and degree ofthe color transition. In accordance with an important feature of thepresent invention, an indicator reagent composition of the presentinvention provides a sufficiently resolved and differentiated colortransition such that the total available chlorine in a test sample canbe measured and accurately determined without the use of color-measuringinstruments, such as spectrophotometers or calorimeters, over aconcentration range of 0 to greater than 5000 ppm. However, if desired,such color-measuring instruments can be used to measure the differencein color degree and intensity between the test sample and a solutionhaving a known concentration of total available chlorine.

The intensity and degree of the color transition are used to determinethe total available chlorine content of the test sample by comparing orcorrelating the color produced by the test sample to colors produced bysolutions having a known total available chlorine concentration. Inaccordance with an important feature of the present invention, theindicator reagent composition provides a sufficiently resolved anddifferentiated color transition such that the total available chlorineof the test sample can be measured for test samples having a totalavailable chlorine content of 0 to greater than about 5000 ppm withoutthe use of color-measuring instruments.

An indicator reagent composition of the present invention, as describedabove, is used in dry phase, test pad assays for total availablechlorine. The dry phase, test pad assay for total available chlorineutilizing a present indicator reagent composition is performed inaccordance with methods well known in the art. In general, the assay fortotal available chlorine is performed by contacting the test sample withan analyte detection device that includes an indicator reagentcomposition. The analyte detection device can be dipped into the testsample, or the test sample can be applied to the analyte detectiondevice dropwise. As set forth hereafter, by applying the test sample tothe localized area of the detection device, such as from a fine-tippedpipette or syringe needle, the relative amounts of free and boundavailable chlorine in the test sample can be determined.

The resulting change in color of the analyte detection device revealsthe total available chlorine concentration of the test sample; and, ifso designed, the resulting color transition can be compared to astandardized color chart to provide a measurement of the total availablechlorine concentration of the test sample.

Typically, the analyte detection device is a test strip impregnated withan indicator reagent composition, designed either as a single pad teststrip (to assay only for a single analyte) or as a multiple pad teststrip (to assay for several analytes simultaneously). For either type oftest strip, the test strip includes a support strip, or handle, normallyconstructed from a hydrophobic plastic, and a reagent test pad,comprising a bibulous or nonbibulous carrier matrix. In general, thecarrier matrix is an absorbent material that allows the test sample tomove in response to capillary forces through the matrix to contact theindicator reagent composition and produce a detectable and measurablecolor transition.

The carrier matrix can be any substance capable of incorporating thechemical reagents required to perform the assay of interest, as long asthe carrier matrix is substantially inert with respect to the chemicalreagents. The carrier matrix also is porous or absorbent relative to theliquid test sample.

The expression "carrier matrix" refers either to bibulous or nonbibulousmatrices that are insoluble in the carrier of the indicator reagentcomposition and other physiological fluids and that maintain theirstructural integrity when exposed to the carrier and other physiologicalfluids. Suitable bibulous matrices include filter paper, spongematerials, cellulose, wood, woven and nonwoven fabrics, and the like.Nonbibulous matrices include glass fiber, polymeric films, andmicroporous membranes. Other suitable carrier matrices includehydrophilic inorganic powders, such as silica gel, alumina, diatomaceousearth and the like; argillaceous substances; cloth; hydrophilic naturalpolymeric materials, particularly cellulosic material, like cellulosebeads, and especially fiber-containing papers such as filter paper orchromatographic paper; synthetic or modified naturally occurringpolymers, such as cellulose acetate, polyvinyl chloride, polyacrylamide,polyacrylates, polyurethanes, crosslinked dextran, agarose, and othersuch crosslinked and noncrosslinked water-insoluble hydrophilicpolymers. The carrier matrix can be of different chemical compositionsor a mixture of chemical compositions. The matrix also can vary inregards to smoothness and roughness combined with hardness and softness.The handle usually is formed from hydrophobic materials such ascellulose acetate, polyethylene terephthalate, polycarbonate, orpolystyrene, and the carrier matrix is most advantageously constructedfrom filter paper of polymeric films.

The carrier matrix of the test strip can be any bibulous or nonbibulousmaterial that allows permeation by the test sample to saturate the testpad of the test strip that is impregnated with the indicator reagentcomposition. A preferred carrier matrix is a hydrophilic, bibulousmatrix, including cellulosic materials, such as paper, and preferablyfilter paper. The carrier matrix also can be a hydrophilic, nonbibulousmatrix, including polymeric films, such as a polyurethane or acrosslinked gelatin. Such polymeric films possess all of the qualitiesrequired of a carrier matrix of the present invention, includingsuspending and positioning both the essential ingredients and anyoptional ingredients included in the indicator reagent composition, andpermeability of the test sample through the carrier matrix.

In accordance with the method of the present invention, to perform a dryphase test strip assay for total available chlorine, an acetonesolution, including: (a) about 1 to about 200 mM of an indicator, suchas tetramethylbenzidene; (b) about 0.05% to about 1.5% by weight of asurfactant, like a sulfosuccinate; (c) 0% to about 4% by weight of apolymer, like a cellulose-type polymer; and (d) any other desiredoptional ingredients, or solvents, first is prepared. A nonbibulousmatrix, such as a polyurethane film, or a bibulous matrix, such asfilter paper, then is saturated or impregnated with the acetone solutionby immersing or by spraying the acetone solution onto sheets or precutstrips or pads of the polyurethane film or filter paper.

Then, after removing the acetone solvent by drying in a forced air ovenat a temperature of about 40° C. to about 10° C. for about 2 minutes toabout 5 minutes, the polyurethane film or filter paper is saturated andimpregnated with an aqueous solution, including: (a) about 20 to about600 mM of a buffer, like a citrate buffer; (b) 0 to 1000 ppm of acatalyst, like a peroxidase or iodide ion; (c) 0% to about 4% by weightof a polymer; and (d) any other desired optional ingredients,surfactants, or solvents, like background dyes, either by immersion orby spraying. After a second oven drying at about 40° C. to about 100° C.for approximately 2 minutes to 15 minutes, the twice-saturated ortwice-impregnated polyurethane film or filter paper, if necessary, iscut to an appropriate size, such as a pad having dimensions of about 0.2in. (inch) (0.5 cm) by about 0.5 in (1.3 cm) to about 0.5 in. (1.3 cm)by about 1 in. (2.5 cm).

It should be understood that it is well within the experimentaltechniques of those skilled in the art of preparing test devices todetermine the proper balance between size of the test pad, the strengthof indicator reagent composition solutions, the amount of test sample,and the method of introducing the test sample to the test strip, such asby pipetting rather than dipping, in order to design a quantitativeassay for total available chlorine utilizing the method and compositionof the present invention.

The dried, twice-impregnated polyurethane film or filter paper then issecured to an opaque or transparent hydrophobic plastic handle withdoublesided adhesive tape. The resulting test strip then is contactedwith a test sample for a sufficient time to saturate the test pad withthe sample. After waiting a predetermined time, such as from about 1second to about 120 seconds, the test strip is examined, either visuallyor by instrument, for a response. The color transition, if any, of thetest pad reveals the concentration of total available chlorine in thetest sample.

In many cases, simple visual observation of the test strip provides thedesired information. If more accurate information is required, a colorchart bearing color spots corresponding to various known concentrationsof total available chlorine can be prepared for the particular indicatorreagent composition used in the test strip. The resulting color of thetest strip after contact with the test sample then can be compared withthe color spots on the chart to determine the concentration of totalavailable chlorine in the test sample. If a still more accuratedetermination is required, a spectrophotometer or calorimeter can beused to more precisely determine the degree of the color transition. Inaddition, the dry phase test strip assay can be made quantitative byemploying spectrophotometric or calorimetric techniques, as opposed tovisual techniques, in order to more reliably and more accurately measurethe degree of color transition, and, therefore, more accurately measurethe concentration of total available chlorine in the test sample.

In accordance with one embodiment of the present invention, thefollowing dry phase test strips were prepared to perform a dry phaseassay for total available chlorine. A strip, a pad, or a sheet of acarrier matrix, like filter paper, first was immersed into an acetonesolution including:

INDICATOR REAGENT COMPOSITION Formulation

    ______________________________________    First Immersion Solution    Ingredient            Amount    ______________________________________    Acetone               40     g    TMB.sup.1)            0.5    g    10% KLUCEL.sup.2)     5      g    Surfactant.sup.3)     0.02   g    ______________________________________     .sup.1) tetramethylbenzidine indicator;     .sup.2) a 10% aqueous solution of hydroxypropylcellulose, KLUCEL is     available from Aqualon Co., Wilmington, DE; and     .sup.3) AEROSOL OT, dioctyl sodium sulfosuccinate, available from Cytec     Industries, West Paterson, NJ.

Excess solution was removed from the surface of the filter paper with ascraper bar.

The once-saturated or impregnated filter paper then was dried in aforced air oven having a temperature of about 45° C. to about 80° C. forabout 5 minutes. After drying, the once-saturated or impregnated filterpaper then was immersed into an aqueous solution including:

    ______________________________________    Second Immersion Solution    Ingredient              Amount    ______________________________________    Water                   30    g    Citrate Buffer (1M) (pH 5.1)                            3     g    Peroxidase              30    mg    2.5% NATROSOL.sup.4)    1.5   g    2% BENECEL.sup.5)       3     g    ______________________________________     .sup.4) a 2.5% aqueous solution of hydroxyethylcellulose, NATROSOL is     available from Aqualon Co.; and     .sup.5) a 2% aqueous solution of hydroxyethylmethylcellulose, BENECEL is     available from Aqualon Co.

The twice-saturated or impregnated filter paper then was dried in anoven having a temperature of about 40° C. to about 80° C. for about 5minutes. The dried and twice-saturated or impregnated filter paper thenwas backed with a double-sided adhesive, and slit into 0.2 inch (0.5 cm)wide ribbons. A ribbon of filter paper incorporating an indicatorreagent composition of the present invention then is attached to apolystyrene plastic support by means of the double-sided adhesive. Theplastic support, including the saturated or impregnated filter paper,then is slit into 0.2 inch (0.5 cm) wide strips. Accordingly, theplastic support includes a pad having dimensions of about 0.2 inch (0.5cm) by about 0.2 inch (0.5 cm) of saturated or impregnated filter paperto provide a test pad comprising a filter paper carrier matrixincorporating an indicator reagent composition of the present invention.

In addition, it should be understood that an indicator reagentcomposition of the present invention demonstrates sufficient stabilitysuch that the carrier matrix can be saturated or impregnated byimmersing the carrier matrix into a single aqueous solution, or a singleaqueous acetone solution, including all of the essential and optionalingredients of the indicator reagent composition. However, the two-stepmethod utilizing two immersions is preferred because certain indicatorreagent composition ingredients have relatively low water solubilities,and a more stable color transition is observed.

To demonstrate the new and unexpected results achieved by the method ofthe present invention, dry phase test strips incorporating an indicatorreagent composition of the present invention (Formulation #1) were usedto assay standardized solutions containing available chlorine.Individual test strips were dipped into a series of standardizedsolutions, containing from 0.5 ppm to 5000 ppm available chlorine. Thestandardized solutions were prepared by diluting a 5.25% (by weight)sodium hypochlorite solution with water.

The standardized solutions were assayed for total available chlorine bycontacting the test strip with a standardized solution for about one (1)second. The color of the test strips then was observed. Timing of thestrip reaction is not critical. However, for consistency, the stripcolor was evaluated after 60 seconds in each test. It should be notedthat the present test strips can detect 0.1 ppm or less total availablechlorine by contacting the test strip with the test sample for 60seconds, or can detect 0.05 ppm or less total available chlorine bycontacting the test strip with the test sample for 120 seconds. Anytrace of a blue color is considered a positive test for availablechlorine. The test results are set forth in Table 1.

                                      TABLE 1    __________________________________________________________________________    Dilution of 5.25%              1/10                  1/20                     1/50                        1/100                           1/200                              1/500                                 1/1000                                     1/5000                                         1/10,000                                              1/50,000                                                   1/100,000    Sodium Hypochlorite    Solution.sup.6)    ppm of Available              5000                  2500                     1000                        500                           250                              100                                 50  10  5    1    0.5    Chlorine    Color Transition.sup.7)              Brown                  Black                     Dark                        Deep                           Navy                              Sky                                 Sky Aqua                                         Light                                              Lighter                                                   Positive                     Blue                        Blue                           Blue                              Blue                                 Blue                                     Blue                                         Blue Blue Blue    __________________________________________________________________________     .sup.6) diluting 1 volume part of aqueous 5.25% (by weight) sodium     hypochlorite solution with indicated volume parts of water; and     .sup.7) test strip was colorless prior to immersion into the standardized     solution.

The results set forth in Table 1 show that a test strip of the presentinvention is capable of assaying for total available chlorine over theentire range of 0 to greater than 5000 ppm by providing a differentiablecolor response over this entire range. Accordingly, a single test stripcan be used to ensure that a sanitizing solution has sufficient strengthto perform its intended function, and that the residual chlorine (orresidual peroxide) in rinse water is below toxic levels.

In particular, Table 1 shows that if the test strip shows any degree ofblue color, then the rinse water contains a potentially toxic amount offree available chlorine, and, therefore, rinsing of the hemodialysisunit with deionized water should be continued. Similarly, if that sametest strip is dark blue to black to brown, then the solution beingpumped through the hemodialysis unit contains a sufficient amount offree available chlorine to sanitize the unit. The intermediate valuesfor total available chlorine are important in other applications, forexample, in restaurant sanitizing solutions or in swimming poolsapplications, to ensure that proper levels of total available chlorineare present.

The results in Table 1 also show that in the low concentration range(e.g., about 500 ppm or less) color differentiation between differenttotal available chlorine concentrations is relatively easy todistinguish. The color transitions in the high range are particularlyuseful in ensuring that hemodialysis units are properly cleaned betweenpatients because the sanitizing solution needed to clean thehemodialysis unit requires a free available chlorine concentration of atleast 500 ppm.

In other tests, the affect of a surfactant on the ability of anindicator reagent composition of the present invention to assay over abroad total available chlorine range also was investigated. Aspreviously stated, it is desirable to extend an assay for totalavailable chlorine to include a high detection range of 1000 ppm orgreater. This was difficult to achieve using prior test strips unlessthe test sample was diluted to reduce the chlorine concentration towithin the detectable range of the test strip. Test sample dilution isundesirable because of the time involved and because of the distinctpossibility is of dilution error, and, in turn, assay error.

An assay having a broad total available chlorine detection range allowsthe user to directly monitor, without dilution, the effective chlorinelevel of a sanitizing or disinfecting solution. In particular, a 1:10dilution of a 5.25% sodium hypochlorite solution with water, whichcommonly is used in hemodialysis operations to sanitize the dialysissystem, can be assayed using a present test strip. The problem withcurrently available test strips is that the detection ranges are narrowand confined to a specific range, typically 0 to 10 ppm, although somestarch-iodine papers have a detection range of 10 to 200 ppm. In allcases, the detection range is confined to low or middle concentrationrange, and no present test strip has a detection range to assay for bothlow and middle concentration levels of total available chlorine, letalone a high concentration level of total available chlorine.

For example, when tetramethylbenzidene (TMB) is used as the indicator,the oxidized, blue form of TMB results in test samples having a low freeavailable chlorine concentration. This blue form of oxidized TMB quicklyconverts into a higher oxidation state, and becomes yellow/brown incolor. It has been found that surfactants, and especially anionicsurfactants, prevent such a conversion, and, accordingly, the browncolor form of oxidized TMB is formed only at much higher free availablechlorine concentrations.

To demonstrate the effect of a surfactant, solutions containingdifferent surfactants were individually impregnated onto filter paper,such as Schleicher & Schuell 903. The solutions contained the followingingredients, wherein the identity of the surfactant was varied:

    ______________________________________    Acetone                5      g    Tetramethylbenzidene   0.5    g    Surfactant             0.005  g.    ______________________________________

Filter paper was dipped in a solution, then dried at 65° C. for 5minutes. The dried filter papers were made into strips by cutting into0.2 inch squares, which were adhered to a plastic handle usingdouble-side adhesive tape. The resulting test strips were dipped intosolutions having different free available chlorine concentrations. Thecolor of each test strip was visually observed and recorded. Table 2summarizes the test results using different surfactants.

                  TABLE 2    ______________________________________    Effect of Surfactant on Assay Range Using Tetramethylbenzidine    Dilution    Factor.sup.6)              1:10    1:50    1:500 1:5000 1:10,000    ______________________________________    Nonionic    Surfactants.sup.8)    BRIJ 35   Y.sup.12)                      Y       Y/Gr  Bl     Lt/Blue    TWEEN 20  Y       Y       Y/Gr  Bl     Lt/Blue    TRITON X-100              Y       Y       Y/Gr  Bl     Lt/Blue    Anionic    Surfactants.sup.9)    DBS       Br      Bk/Bl   Bl    Bl     Lt/Blue    SDS       Br      Bk/Bl   Bl    Bl     Lt/Blue    DOSS      Br      Gr/Bl   Bl    Bl     Lt/Blue    Cholate   Br      Br      Bl    Bl     Lt/Blue    Benzoic acid.sup.10)              Br      Bk/Bl   Bl    Bl     Lt/Blue    Lauric acid              Br      Bk/Bl   Bl    Bl     Lt/BIue    Caprylic acid              Br      Bk/Bl   Bl    Bl     Lt/Blue    Cationic    Surfactants.sup.11)    CTAB      Y       Y       Gr    Gr     Gr    CPC       Y       Y       Y     Bl     Lt/Blue    Zwitterionic    Surfactants.sup.11)    Z-08      Y       Y       Gr    Bl     Lt/Blue    CHAPS     Y       Y       Gr    Gr     Lt/Blue    Control    None      Y       Y       Gr    Gr     Lt/Blue    ______________________________________     .sup.8) BRIJ 35polyethylene glycol dodecyl ether (23 moles ethylene     oxide), TWEEN 20polyethylene glycol sorbitan monolaurate (20 moles     ethylene oxide), and TRITON X100-polyethylene glycol tertoctyl phenyl     ether (9 moles ethylene oxide);     .sup.9) DBSdodecylbenzene sulfonate, SDSsodium dodecylsulfate, DOSSdiocty     sulfosuccininate, and cholatesodium salt of cholic acid;     .sup.10) benzoic acid is not a surfactant, but is capable of stabilizing     color formation;     .sup.11) CTABcetyltrimethylammonium chloride, CPCcetylpyridinum chloride,     Z08-Zwittergent 308, and     CHAPS3, (3cholamidopropyl)dimethylammonio1-propane sulfonate; and     .sup.12) color designations: Yyellow; Brbrown; Bkblack; Dkdark; Grgreen;     Blblue; Y/Gryellowish green; Bk/Blblack blue, Dk/Bldark blue, Lt/Bllight     blue.

As illustrated in Table 2, anionic surfactants have an excellent abilityto prevent oxidized tetramethylbenzidine, that is blue in color, frombeing further oxidized and turning brown in color. The class of anionicsurfactant present in an indicator reagent composition is not important,e.g, sulfate, sulfonate, carboxylate, phosphate, and similar hydrophilicmoieties are useful. However, the hydrophobic moiety of the anionicsurfactant can have an effect. The tests show that a linear hydrophobicmoiety, alone or with phenyl groups, is slightly more effective instabilizing the color of the indicator than a bulkier hydrophobicmoiety, such as cholate or dioctyl sulfosuccinate. It is theorized thatboth the hydrophilic group, and the hydrophobic moiety of the surfactantinteract with the indicator, thereby protecting the indicator fromfurther oxidation, and further color change. Nonionic surfactants alsowere effective in stabilizing the color transition, but cationic andzwitterionic surfactants failed to effectively prevent further oxidationof an oxidized indicator.

As previously stated, the indicator reagent composition is buffered in apH range of about 4 to 5 about 6. It also has been found that theidentity of the buffer has an effect with respect to stabilizing thecolor transition and preventing the colored, oxidized form of theindicator from being further oxidized and turning the test strip brown.In particular, a citrate buffer and a phosphate buffer were equallyeffective with respect to stabilizing the color transition of a TMBindicator in the presence of free available chlorine, although the colorof a test strip containing a citrate buffer has a brighter and cleanerblue color than a test strip containing a phosphate buffer. Thefollowing Table 3 illustrates the effect of different buffers on colorstability of an indicator reagent composition using TMB as theindicator.

                  TABLE 3    ______________________________________    Effect of Buffer on Color Stability    Dilution Factor.sup.6)                1:10    1:50    1:500 1:5000                                            1:10,000    ______________________________________    Buffer (pH)    Citrate (5.1)                Br.sup.12)                        Bk/Bl   Bl    Bl    Lt/Bl    Succinate (5.2)                Br      Bk/Bl   Bl    Bl    Lt/Bl    2-Ketoglutarate (5.6)                Br      Br      Bl    Bl    Lt/Bl    Lactate (5.1)                Br      Dk/Bl   Bl    Bl    Lt/Bl    Phosphate (5.3)                Br      Bk/Bl   Bl    Bl    Lt/Bl    Borate (5.4)                Y       Y       Gr    Lt/Bl Lt/Bl    ______________________________________

In addition, the ability of a peroxidase to stabilize the colortransition of a test strip and catalyze the assay for total availablechlorine was demonstrated by adding 50 milligrams of horseradishperoxidase to the second immersion solution of Formilation #1, preparingtest strips, and using the test strips to assay for total availablechlorine of solutions containing chloramine. The tests showed thatperoxidase increased the reaction rate to convert bound chlorine to freechlorine, and the test strip underwent a complete color transitionfaster than a test strip lacking peroxidase.

From the visual assays and the data presented in Tables 1-3, it has beendemonstrated that a particularly useful indicator istetramethylbenzldine. An indicator reagent composition of the presentinvention that includes tetramethylbenzidine, in addition to asurfactant and a buffer to buffer the composition to a pEi of about 4 toabout 6, exhibits a sufficiently dramatic color transition, from lightblue to brown, to provide a sensitive and accurate assay for totalavailable chlorine in a test sample. The color transition also issufficiently resolvable and differentiable, either visually or byinstrument, such that an unknown concentration of total availablechlorine in a test sample can be determined. Furthermore, it has beenfound that the indicator is stabilized by interaction with a surfactant,and that a catalyst can convert bound available chlorine to freeavailable chlorine, such that the color transition endpoint is reachedwithin about 1 second to about 2 minutes.

In accordance with an important feature of the present invention, it hasbeen found that a present indicator reagent composition can be used tosimultaneously assay for free and bound available chlorine on a singletest strip. In particular, it is desirable to utilize a single indicatorreagent composition to react with two or more analytes in a singlesample in such a way that the analytes can be either individually orsimultaneously detected. In the treatment of municipal water, swimmingpool water, or sanitizing solutions, an assay for the different forms ofchlorine in water, i.e., free or bound available chlorine, and therelative amount of these forms of chlorine, provides importantinformation with respect to the effectiveness and safety of chlorinesolution.

In particular, the prior examples assayed for total available chlorineby dipping a test strip impregnated with the composition of Formulation#1 into a test sample. It has been found that by applying the testsample to a test strip from a fine-tipped pipette or a syringe needlepermits the simultaneous detection of free and bound available chlorinein the test sample.

This method of application applies the test sample to a localized areaof the test strip, in the form of a dot. Typically, a sufficient amountof test sample is applied from a fine-tipped pipette or syringe needleto the test device to provide a circular area, or dot, having a diameterof about 1 to about 2 mm (millimeters). The test sample can be appliedin a sufficient amount, for example, from about a 20- to about a27-gauge syringe needle.

Tests showed that by applying a solution of free chlorine onto the teststrip using a needle, as opposed to dipping, a blue dot formedessentially immediately at the zone of sample application. Very littleto no color formation was observed on the area surrounding the blue dotat the zone of application. This test is illustrated in FIG. 1. However,when a chioramine solution was applied to a test strip from a needle inthe same manner, a uniform blue color was observed across the entiretest pad. This test illustrated in FIG. 2. Finally, when a solutioncontaining both free chlorine and chloramine was applied onto a teststrip from a syringe needle, a blue dot at the zone of application andblue background color across the entire strip was observed.

Because free chlorine quickly reacts with the indicator reagentcomposition, a concentrated blue dot at the area of test sampleapplication to the test pad is formed. Chloramine, however, is a slowreacting form of chlorine that reacts through a mediator compound.Therefore, the blue color attributed to bound chlorine spreadsthroughout the test pad. Because distinct color patterns are generatedfrom test samples having different forms of chlorine present, asimultaneous detection of one or both types of chlorine species occurs.

In addition, color intensities of the blue dot and the blue-colored testpad are related to the relative amount of free chlorine and chloraminein the test sample. The blue-colored pattern on the test pad provides aqualitative assay for the type of chlorine in the test sample, and therelative intensity of the blue dot and blue background color provides aquantitative assay for the ratio of the chlorine species in the testsample. This quantitative assay can be achieved by comparing the colorof the blue dot and the surrounding blue area to standardized colorcharts determined from known free and bound chlorine concentrations.

In accordance with an important feature of the present invention, thecontinuing and substantial problems in dry phase test strips forassaying a wide concentration range of total available chlorine,including the instability of the indicator, are essentially eliminatedby the present invention. An indicator reagent composition of thepresent invention provides a differentiable response to total availablechlorine over the concentration range of 0 to greater than 5000 ppm ofthe test sample. Therefore, accurate and reliable assays for totalavailable chlorine in test samples can be performed by utilizing anindicator reagent composition and device of the present invention. Thetest strips also can be utilized to simultaneously assay for thepresence, and the relative amounts, of free and bound available chlorinein a test strip.

Obviously, many modifications and variations of the invention ashereinbefore set forth can be made without departing from the spirit andscope thereof, and, therefore, only such limitations should be imposedas are indicated by the appended claims.

What is claimed is:
 1. A method of determining free available chlorineand bound available chlorine content of an aqueous sample, said methodcomprising:(a) applying the aqueous sample to a localized area of a testpad, said test pad having incorporated therein an indicator reagentcomposition, wherein said composition comprises:(i) an indicator capableof interacting with free available chlorine, (ii) a buffer, (iii) anonionic surfactant, an anionic surfactant, or a mixture thereof, and(iv) a carrier comprising water, wherein the composition has a pH ofabout 4 to about 6; (b) determining the free available chlorine contentof the aqueous sample from the intensity and degree of a colortransition of the indicator reagent composition at the localized area ofthe test pad; and (c) determining the bound available chlorine contentof the aqueous sample from the intensity and degree of a colortransition of the indicator reagent composition at an area of the testpad outside of the localized area.
 2. The method of claim 1 wherein theindicator reagent composition further comprises:(v) a catalyst selectedfrom the group consisting of a peroxidase enzyme, iodide ion, andmixtures thereof; and (vi) a polymer.
 3. The method of claim 1 whereinthe aqueous test sample has a total available chlorine content of 0 togreater than 5000 ppm.
 4. The method of claim 1 wherein the intensityand degree of the color transitions are determined visually orinstrumentally.
 5. The method of claim 1 wherein the indicator comprisesa redox indicator, a heterocyclic azine indicator, or a mixture thereof.6. The composition of claim 1 wherein the indicator is a benzidine-typeindicator having a structure ##STR3## wherein R¹ and R² substituents,either the same or different, are selected from the group consisting ofhydrogen, a lower alkyl group, a lower alkyloxy group, aryl, andaryloxy, or the R² substituents can be taken together to form .parenopen-st.CH₂ .paren close-st._(n), wherein n is 1 or
 2. 7. The method ofclaim 1 wherein the composition has a pH of about 4.5 to about 5.5. 8.The method of claim 1 wherein the nonionic surfactant has an HLB valueof about 6 to about
 25. 9. The method of claim 2 wherein the catalyst ispresent in an amount of 0 to about 1000 ppm.
 10. The method of claim 2wherein the peroxidase enzyme comprises horseradish peroxidase.
 11. Themethod of claim 2 wherein the iodide ion is present as an alkali metaliodide.
 12. The method of claim 2 wherein the polymer is present in anamount of 0% to about 4% by weight of the composition.
 13. The method ofclaim 2 wherein the polymer is selected from the group consisting ofpolyvinylpyrrolidone, an ester of a PVM/MA copolymer, polyvinyl alcohol,gum arabic, gelatin, algin, carrageenan, casein, albumin, methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose,hydroxybutylcellulose, sodium carboxymethylcellulose, and mixturesthereof.
 14. The method of claim 1 wherein the composition comprises:(a)about 10 to about 150 millimoles per liter of the composition of abenzidine-type indicator; (b) about 50 to about 200 millimoles per literof the composition of a buffer selected from the group consisting of apolycarboxylic acid having carboxyl groups separated by two to fivecarbon atoms; (c) about 0.1 to about 1% by weight of the composition ofan anionic surfactant; and (d) a carrier comprising water, wherein thecomposition has a pH of about 4.8 to about 5.3.
 15. The method of claim14 where the composition further comprises:(e) 0 to about 10 ppm iodideion, 0 to about 1000 ppm peroxidase enzyme, or a mixture thereof; and(f) 0% to about 3% by weight of the composition of a cellulose-typepolymer.
 16. The method of claim 1 wherein the aqueous sample is appliedto the test pad from a pipette or a syringe needle.
 17. A method ofsimultaneously determining the free available chlorine and boundavailable chlorine content of an aqueous sample comprising:(a) applyingthe aqueous sample to a localized area of a test pad of an analytedetection device, said a test pad having incorporated therein:(i) anindicator capable of interacting with free available chlorine, (ii) abuffer to maintain a pH of about 4 to about 6, (iii) a nonionicsurfactant, an anionic surfactant, or a mixture thereof, and (iv) acarrier comprising water; and (b) examining the test pad for colortransitions; (c) correlating a color transition of the localized area tothe free available chlorine content of the aqueous sample; and (d)correlating a second color transition of the test pad outside thelocalized area to the bound available chlorine content of the aqueoussample.
 18. The method of claim 17 wherein the indicator is present in aconcentration of about 1 to about 200 millimoles per liter of thecomposition.
 19. The method of claim 17 wherein the indicator is presentin a concentration of about 1 to about 200 millimoles per liter of thecomposition.
 20. The method of claim 17 wherein the buffer is present ina concentration of about 20 to about 600 millimoles per liter of thecomposition.
 21. The method of claim 16 wherein the surfactant ispresent in an amount of about 0.05% to about 1.5% by weight of thecomposition.